Project Summary The efficiency of bacterial killing by alveolar macrophages (AM?) is an essential determinant of the lung's ability to resolve inflammatory lung injury, and indeed may be required for the development of tolerance to lung injury induced by secondary infection. However, the molecular mechanisms of bacterial killing are not well understood. The crucial observation underpinning Project 1 shows a fundamental host-defense function of the ROS-sensitive transient receptor potential melastatin-2, TRPM2, a phagosomal membrane-associated cation channel, in regulating bactericidal activity of M?. TRPM2 was essential for controlling the pH of phagosomes and blocking of the TRPM2-mediated acidification prevented bacterial killing and, moreover, enhanced inflammatory lung injury. Thus, in Project 1 we will test the central hypothesis that phagosome-associated TRPM2 in M? promotes the resolution of inflammatory lung injury by regulating phagosomal acidification, and thereby is a central mechanism for activating bacterial killing. This hypothesis will be tested by addressing the following Specific Aims (SA). SA #1 will determine the role of phagosomal membrane TRPM2 activation induced by the oxidases NOX2 and/or NOX4 in regulating the phagosomal acidification property of M?, and thus in generating bactericidal-competent M?. SA #2 will define the crucial TRPM2-regulated negative feedback mechanism in M? that may also acidify phagosomes through dampening NOX2/NOX4-mediated ROS production, and thus the promote bacteria killing function of M?. SA #3 will determine the role of TRPM2 regulated phagosomal acidification in resolving inflammatory lung injury using genetically modified models with specific deletion of TRPM2 in phagocytic cells, and the role enhanced phagosomal acidification in promoting the tolerance to injury induced by secondary infection. We posit that by identifying the central signaling mechanisms responsible for TRPM2 activation in the M? phagosomes, it will be possible to develop strategies to more effectively resolve inflammatory lung injury and to make lung's tolerance to injury through enhancing bacterial killing function of M?.